More than a quarter of a century after the first AIDS cases were described, the basic immunologic mechanisms that control HIV-1 remain unknown. Induction of HIV-1-specific CD8 T cells is associated with a decline in plasma viremia following acute infection, but neither the magnitude nor breadth of these responses, as measured by IFN-3 expression, is correlated with disease control. These assays do not assess the ability of CTL to recognize and kill virus- infected cells. Indeed, the properties currently measured may simply reflect the level of viremia, rather than reveal causes of immune control. We hypothesize that identifying key antiviral effector functions in CTL, and potential defects in these activities, will require direct examination of the interaction between T cells and live HIV-1-infected cells, which has never been comprehensively analyzed. During previous funding periods of this grant, we developed a robust in vitro viral inhibition assay that allows identification of T cells that control HIV-1 replication. Our data indicate that this short-term co-culture assay readily distinguishes functional and non-functional CTL: even within a single individual, we observed 1,000-fold differences between T cells with differing specificities. This powerful tool allows for detailed cellular and molecular analyses, since each component of the system (CTL, virus, and target cell) can be manipulated individually. We now propose to define CTL populations that inhibit HIV-1 infection in vitro and to directly assess the molecular pathways involved in antiviral control and loss of CTL function. Specifically we propose to: 1) Identify CTL epitope specificities that are best able to control HIV-1 replication in vitro; 2) Determine the impact of TCR and viral sequence variation on CTL avidity, cell signaling, and antiviral inhibition capacity; and 3) Define essential antiviral CTL effector molecules and important regulatory pathways in CTL using gene expression analysis, over-expression vectors, and RNAi technology. These studies are highly relevant to vaccine design and testing since they will provide critical new data to enhance our understanding of antiviral CTL function and they will identify viral regions that are efficiently targeted by host CTL that can be incorporated into novel immunogens.